Inhibitory Effect of Photodynamic Therapy with Indocyanine Green on Rat Smooth Muscle Cells.

BACKGROUND: Vascular smooth muscle cells play a critical role in the intimal hyperplasia of restenosis. A previous study of a rat balloon injury model demonstrated that photodynamic therapy (PDT) using indocyanine green (ICG) and near-infrared (NIR) light irradiation reduced intimal hyperplasia in carotid arteries. However, the effect of ICG-PDT on smooth muscle cells remains unclear. This study aimed to evaluate the effects of PDT with ICG and NIR irradiation on the viability of vascular smooth muscle (A-10) cells. METHODS: A-10 cells were incubated with ICG at different concentrations for different time intervals. Intracellular accumulation of ICG inside the cells was observed by light microscopy, ultraviolet-visible (UV-VIS) spectrophotometry and spectrofluorometry. Cell viability and cell death after ICG-PDT were assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay and lactate dehydrogenase release assay. Changes in nuclear morphology and cell cycle distribution were evaluated to determine the possible cell death mechanism mediated by ICG-PDT. RESULTS: ICG uptake in A-10 cells increased with the amount of ICG in the culture media. The intracellular accumulation of ICG reached a maximum at 8 h. After ICG-PDT, cell viability decreased and cell death increased in a concentration- dependent manner. The half maximal inhibitory concentration of ICG was 8.3 µM with 4 J/cm2 NIR irradiation. Membrane blebbing and chromatin condensation were observed, and the percentage of cells in the sub-G1 phase increased after ICG-PDT. Thus, apoptosis might be responsible for decreasing the viability of A-10 cells by ICG-PDT. CONCLUSIONS: This study demonstrated that ICG-PDT had an inhibitory effect on smooth muscle cells, possibly via an apoptosis pathway.

Photodynamic therapy of balloon-injured rat carotid arteries using indocyanine green.

Photodynamic therapy (PDT) has been used to inhibit intimal hyperplasia in injured arteries. Because of the limited tissue penetration of visible light, an endovascular light source with a guided wire is often required for effective treatment. Indocyanine green (ICG), a near-infrared (NIR) photosensitizer, has been used in PDT for cancers. An extracorporeal light source may be used for shallow tissue because of the better tissue penetration of NIR light. The aim of this study was to evaluate the effect of ICG-PDT using extracorporeal NIR light on the inhibition of intimal hyperplasia in balloon-injured carotid arteries. A balloon injury (BI) model was used to induce intimal hyperplasia of carotid artery. Sprague-Dawley rats were divided into control, BI, BI + 1 × PDT, and BI + 2 × PDT groups. The control group underwent a sham procedure. PDT was performed 7 days after BI. In the BI + 1 × PDT group, ICG was administered 1 h before light irradiation. External illumination with 780-nm light-emitting diode light at a fluence of 4 J/cm2 was applied. For the BI + 2 × PDT group, PDT was performed again at day 7, following the first PDT. Hematoxylin and eosin (H & E) staining was performed to assess vessel morphology. Arterial wall thickness was significantly larger in the BI group compared with the control group. ICG-PDT significantly reduced arterial wall thickness compared with the BI group. Repeated PDT further decreased arterial wall thickness to the level of the control group. These findings indicate a promising approach for the treatment of restenosis of carotid arteries.

Naringenin more effectively inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in macrophages than in microglia.

Macrophages and microglia are thought to account for initial disease progression in acute myocardial infarction and acute ischemic stroke. Before our study, the inhibitory effects of naringenin, a flavonoid, on lipopolysaccharide (LPS)-induced inflammation in macrophages and microglia have not been fully reported and compared. We hypothesized that naringenin can effectively inhibit LPS-induced inflammation of macrophages and microglia at different concentrations, the range of which is broader, with the lowest concentration more easily achieved in macrophages. In this study, we compared the anti-inflammatory effects of naringenin on LPS-stimulated RAW 274.6 macrophages and BV2 microglia and the suppression effects of naringenin and vitamin C (a well-known anti-inflammatory agent) on LPS-induced nitrite production. The results show that macrophages could maintain cell viability at higher naringenin concentrations and were more easily activated by LPS in comparison to microglia (200 vs 100 µmol/L; 0.1 vs 1 µg/mL). Under LPS (1 µg/mL) stimulation in both cell types, naringenin (up to 200 µmol/L in macrophages and 100 µmol/L in microglia) inhibited nitrite production and inducible nitric oxide synthase and cyclooxygenase-2 expression in a dose-dependent manner. The range of naringenin concentrations for inhibition was broader, and the lowest concentration was more easily achieved in macrophages; the lowest effective concentrations of naringenin to achieve constant suppression effect were 50 µmol/L in macrophages and 100 µmol/L in microglia, respectively. Vitamin C (100 µmol/L), compared with naringenin (100 µmol/L), had less and no suppression effect on LPS (1 µg/mL)-induced nitrite production in macrophages and microglia, respectively. In conclusion, naringenin more effectively inhibits the LPS-induced inflammatory status, including nitrite production and inducible nitric oxide synthase and cyclooxygenase-2 expression, in macrophages than in microglia. The findings of the present study suggest that consumption of naringenin-containing flavonoids might be beneficial to the cardiovascular and cerebrovascular inflammatory process.

Hypoxic preconditioning protects rat hearts against ischaemia-reperfusion injury: role of erythropoietin on progenitor cell mobilization.

Preconditioning, such as by brief hypoxic exposure, has been shown to protect hearts against severe ischaemia. Here we hypothesized that hypoxic preconditioning (HPC) protects injured hearts by mobilizing the circulating progenitor cells. Ischaemia-reperfusion (IR) injury was induced by left coronary ligation and release in rats kept in room air or preconditioned with 10% oxygen for 6 weeks. To study the role of erythropoietin (EPO), another HPC + IR group was given an EPO receptor (EPOR) antibody via a subcutaneous mini-osmotic pump 3 weeks before IR induction. HPC alone gradually increased haematocrit, cardiac and plasma EPO, and cardiac vascular endothelial growth factor (VEGF) only in the first two weeks. HPC improved heart contractility, reduced ischaemic injury, and maintained EPO and EPOR levels in the infarct tissues of IR hearts, but had no significant effect on VEGF. Interestingly, the number of CD34(+)CXCR4(+) cells in the peripheral blood and their expression in HPC-treated hearts was higher than in control. Preconditioning up-regulated cardiac expression of stromal derived factor-1 (SDF-1) and prevented its IR-induced reduction. The EPOR antibody abolished HPC-mediated functional recovery, and reduced SDF-1, CXCR4 and CD34 expression in IR hearts, as well as the number of CD34(+)CXCR4(+) cells in blood. The specificity of neutralizing antibody was confirmed in an H9c2 culture system. In conclusion, exposure of rats to moderate hypoxia leads to an increase in progenitor cells in the heart and circulation. This effect is dependent on EPO, which induces cell homing by increased SDF-1/CXCR4 and reduces the heart susceptibly to IR injury.